As a method for cultivation, there are several methods such as to cultivate by adhering a cell to a carrier or to cultivate a cell by floating in medium. In the case to culture a cell by adhering to a carrier, a material of carrier is an important factor to improve the usefulness for high density and efficient culture regenerative medicine.
As a material for a culture carrier, gelatin, cellulose, hydroxy apatite or collagen are known, however from the view point of practical use for regenerative medicine, it is desirable that a compound is easily absorbed in vivo after embedded. In the case of cellulose, it has a problem that it can not be absorbed in vivo, and from this point of view, collagen attracts attention. Further, as a material of carrier, it is necessary to be investigated from adhering ability of cell and efficiency, and collagen is attracted attention from this point of view.
As a shape of carrier, it is necessary to investigate from macroscopic view point such as stable preservative ability when the carrier is embedded in vivo, or from bioactivity view point such as adhering ability of cell, bioabsorption•effective bio tissue replacement are important factors. As a beads shape carrier, for example, a carrier of agarose beads to which collagen is coated is known, however, it has a problem that the holding of carrier to embedded part is difficult and abilities of absorbable•effective tissue replacement in vivo are not so good. In addition, as a carrier for adhering cell, micro carrier, hollow fiber, membrane or nonwoven cloth are known, and these subjects are improved from holding ability of carrier, however, is not sufficient from said bio activity view point.
In the meanwhile, in recent development of regenerative medicine, it becomes popular to use auto or allo cell or cell of other person for medical treatment. For example, cultivated skin or cultivated cartilage to which only epithelium cell in vitro cultivated or fibroblast cell in vitro that produces interstitial compound in vivo are introduced are developed and is the stage of practical use.
In these regenerative medicines, after cells are cultivated by suitable carrier, it is clearly understood that the use of a cultivated cell which can be put back in vivo together with the carrier is desirable.
Further, as a carrier having said desired characteristic for regenerative medicine, it is considered that such subject can be accomplished by combining a cell, a carrier for cell and an activated factor to promote effective cultivation of cell. For example, in a case of bone or cartilage transplantation, it is important that it further contains sufficiently a matrix component (interstitial compound), such as, apatite, collagen or mucopolysaccharide and others. Bone or cartilage is composed of apatite, collagen, mucopolysaccharide or others which is produced by cell, and as a transplantation fragment for these tissue, it is necessary to contain sufficient amount of matrix component and to have physical characteristics similar in vivo.
Furthermore, because it replaces with own tissue in vivo, matrix components become very important for the purpose to act as an excellent scaffold for cultivation of own cell. In such a requirement, a spongy carrier is proposed (JP62-502936 publication; WO86/05811, document 1), because a spongy carrier of collagen having average pore size of approximately 1 μm to 150 μm improves cultivation efficiency. And, the invention of transplantable transplantation fragment, comprising a carrier with stable three dimensional structure having inner pore which can pass through nutrient liquid and containing cell in it is proposed (JPH8-511679 publication; WO94/20151, document 2), invention of an implantable material produced from a polymer filament having good shape holding ability (JPH9-182784 publication, document 3), further, the invention of matrix for reproduction of cartilage tissue containing collagen II fiber that composes extraceller matrix having good reproduction ability of cartilage tissue when embedded in vivo (JPH11-503338 publication; WO96/25962, document 4) are proposed.
Further, as a collagen carrier for cell cultivation that can cultivate cells by high density and a method for production thereof, a method for production of cell cultivation collagen carrier comprising, having pore size controlled in 50-2000 μm range, said pore is penetrating straightly from one surface to another surface and forming collagen fibrils arranged straightly from one surface to another surface by exposing cell cultivation collagen in which each pores are substantially and independently existing, and by exposing acidic solution to ammonia gas, forming collagen fiber sequenced from one surface to another surface and simultaneously forms straight water pole from one surface to another surface, then evaporate water in gel by freeze dry method is proposed (Japanese Patent 3170693 registered on Mar. 23, 2001, published on May 28, 2001, document 5). And as the effect thereof, following point is mentioned. That is, pore size can be controlled by concentration of ammonia gas and collagen carrier having most suitable pore size and structure to a cell to be cultivated and said pores are controlled along with supplying direction of ammonia gas can be obtained, further surface area of it can be enhanced, therefore, a cell cultivation collagen carrier that can cultivate cells by high density.
Up to this time, an investigation of a carrier as a scaffold of cell was carried out, however, a report of real carrier for regenerative medicine aiming self accumulation of matrix component produced from one's own cell is not found out.
Further, as a factor for activation of cell to said regenerative medicine, factors such as BMP (Bone Morphogeneic Protein), β FGF (β fibroblast growth factor) are known, and recently, recombinant human these factors can use. Further, these factors can be extracted from animal's bone such as bovine and can be used. However, any kind of activation factor, it is important for formation of tissue based on said activation factor to maintain said activation factor by certain concentration at surrounding part.
Recently, big expectation is loaded on regenerative medicine, and development relating to regenerative medicine is becoming an important target, and many firms are joining to the development, however, desired carrier for regenerative medicine is not developed yet, and it is a realistic current problem.
The subject of the present invention is to provide a carrier characterizing, after adhering removed cell or tissue thereof to a carrier and cultivating said removed cell or tissue thereof, transforming the carrier to have figure•structure that is possible to be transplanted in vivo without anaplasia of cell and so as the matrix produced by cell to be accumulated sufficiently in own tissue.
For the purpose to dissolve said subject, the inventors of the present invention basically investigated collagen itself, and considered the development of collagen that can form a cell cultivation carrier, and said collagen can form a carrier having figure•structure possible to accumulate matrix sufficiently in own tissue in vivo, namely, collagen having fine pore structure of figure•structure controlled to thickness direction. Based on said consideration, many experiments were carried out by trial and error, and the inventors of the present invention found out that the neutralization-gellation can be more effectively advanced by controlling fine pore size and progressing direction, and, good effect can be accomplished by transforming soluble collagen to a collagen from which aggregated collagen is removed. For example, the inventors of the present invention found out that by progressing to thickness direction by controlling diffusion, a carrier having figure•structure characterizing that fine pores having uniform fine pore diameter are uniformly sequenced can be obtained, and in said figure•structure, matrix can be sufficiently accumulated in own tissue in vivo, and accomplished to dissolve above mentioned subject.